Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
23 10 2020
23 10 2020
Historique:
received:
26
04
2020
accepted:
02
10
2020
entrez:
24
10
2020
pubmed:
25
10
2020
medline:
11
11
2020
Statut:
epublish
Résumé
Proofreading by replicative DNA polymerases is a fundamental mechanism ensuring DNA replication fidelity. In proofreading, mis-incorporated nucleotides are excised through the 3'-5' exonuclease activity of the DNA polymerase holoenzyme. The exonuclease site is distal from the polymerization site, imposing stringent structural and kinetic requirements for efficient primer strand transfer. Yet, the molecular mechanism of this transfer is not known. Here we employ molecular simulations using recent cryo-EM structures and biochemical analyses to delineate an optimal free energy path connecting the polymerization and exonuclease states of E. coli replicative DNA polymerase Pol III. We identify structures for all intermediates, in which the transitioning primer strand is stabilized by conserved Pol III residues along the fingers, thumb and exonuclease domains. We demonstrate switching kinetics on a tens of milliseconds timescale and unveil a complete pol-to-exo switching mechanism, validated by targeted mutational experiments.
Identifiants
pubmed: 33097731
doi: 10.1038/s41467-020-19165-2
pii: 10.1038/s41467-020-19165-2
pmc: PMC7584608
doi:
Substances chimiques
DNA Primers
0
DNA
9007-49-2
DNA Polymerase III
EC 2.7.7.7
DNA-Directed DNA Polymerase
EC 2.7.7.7
Exonucleases
EC 3.1.-
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
5379Subventions
Organisme : NIGMS NIH HHS
ID : R01 GM110387
Pays : United States
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